Data from human biopsies, in-vitro and in-vivo models, strongly supports the role of thrombin, and its protease-activated receptor (PAR1) in the pathology and progression of glioblastoma (GBM), a high-grade glial tumor. Activation of PAR1 by thrombin stimulates vasogenic edema, tumor adhesion and tumor growth. We here present a novel six amino acid chloromethyl-ketone compound (SIXAC) which specifically inhibits PAR1 proteolytic activation and counteracts the over-activation of PAR1 by tumor generated thrombin. SIXAC effects were demonstrated in-vitro utilizing 3 cell-lines, including the highly malignant CNS-1 cell-line which was also used as a model for GBM in-vivo. The in-vitro effects of SIXAC on proliferation rate, invasion and thrombin activity were measured by XTT, wound healing, colony formation and fluorescent assays, respectively. The effect of SIXAC on GBM in-vivo was assessed by measuring tumor and edema size as quantified by MRI imaging, by survival follow-up and brain histopathology. SIXAC was found in-vitro to inhibit thrombin-activity generated by CNS-1 cells (IC₅₀ = 5 × 10^-11M) and significantly decrease proliferation rate (p < 0.03) invasion (p = 0.02) and colony formation (p = 0.03) of these cells. In the CNS-1 GBM rat animal model SIXAC was found to reduce edema volume ratio (8.8 ± 1.9 vs. 4.9 ± 1, p < 0.04) and increase median survival (16 vs. 18.5 days, p < 0.02 by Log rank Mental-Cox test). These results strengthen the important role of thrombin/PAR1 pathway in glioblastoma progression and suggest SIXAC as a novel therapeutic tool for this fatal disease.

Ehud Sheinberg

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